title: Characterization of a putative stereoselective oxidoreductase from Gluconobacter oxydans and its application in producing ethyl (R)-4-chloro-3-hydroxybutanoate ester.
abstract: A gene encoding an NADH-dependent short-chain dehydrogenase/reductase (gox2036) from Gluconobacter oxydans 621H was cloned and heterogeneously expressed in Escherichia coli. The protein (Gox2036) was purified to homogeneity and biochemically characterized. Gox2036 was a homotetramer with a subunit size of approximately 28 kDa. Gox2036 had a strict requirement for NAD⁺/NADH as the cofactor. Gox2036 displayed preference for oxidation of secondary alcohols and 2,3-diols as well as for reduction of α-diketones, hydroxy ketones, α-ketoesters, and β-ketoesters. However, Gox2036 was poorly active on 1,2-diols and acetoin and showed no activity on primary alcohols, polyols, and aldehydes. The optimum pH values for the oxidation and reduction reactions were 9 and 6, respectively. Gox2036 was highly selective in the reduction of various β-ketones and β-ketoesters. Among the substrates tested, ethyl 4-chloro acetoacetate was reduced to ethyl (R)-4-chloro-3-hydroxybutanoate ester with an excellent conversion yield of 96.9 % and optical purity of >99 % e.e. using an efficient in situ NADH-recycling system involving glucose and a glucose dehydrogenase from Bacillus subtilis (BsGDH).
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, P.O. Box 311#, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Xu
authors.initials: X
authors.lastname: Liu
authors.forename: Rong
authors.initials: R
authors.lastname: Chen
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Yang
authors.forename: Jiale
authors.initials: J
authors.lastname: Wang
authors.forename: Jinping
authors.initials: J
authors.lastname: Lin
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D002087:Butyrates / Q000378:metabolism*; D004926:Escherichia coli; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D002087:Butyrates; C121244:ethyl 4-chloro-3-hydroxybutanoate; D010088:Oxidoreductases